title: The mechanism of Bacillus anthracis intracellular germination requires multiple and highly diverse genetic loci.
abstract: In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.
authors.affiliation: Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: Soumitra
authors.initials: S
authors.lastname: Barua
authors.forename: Matthew
authors.initials: M
authors.lastname: McKevitt
authors.forename: Kevin
authors.initials: K
authors.lastname: DeGiusti
authors.forename: Elaine E
authors.initials: EE
authors.lastname: Hamm
authors.forename: Jason
authors.initials: J
authors.lastname: Larabee
authors.forename: Salika
authors.initials: S
authors.lastname: Shakir
authors.forename: Katie
authors.initials: K
authors.lastname: Bryant
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Koehler
authors.forename: Steven R
authors.initials: SR
authors.lastname: Blanke
authors.forename: David
authors.initials: D
authors.lastname: Dyer
authors.forename: Allison
authors.initials: A
authors.lastname: Gillaspy
authors.forename: Jimmy D
authors.initials: JD
authors.lastname: Ballard
mesh_terms: D000818:Animals; D001408:Bacillus anthracis / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D015169:Colony Count, Microbial; D004251:DNA Transposable Elements; D023061:Gene Order; D005798:Genes, Bacterial; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D016254:Mutagenesis, Insertional; D013171:Spores, Bacterial / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D037521:Virulence Factors